Cytokines that bind the cell surface receptor hek

ABSTRACT

Hek ligand (hek-L) polypeptides as well as DNA sequences, vectors and transformed host cells useful in providing hek-L polypeptides. The hek-L polypeptides bind to a cell surface receptor (hek) that is a member of the receptor tyrosine kinase family. Hek is expressed on cells that include certain tumor cell lines. The hek-L polypeptides also bind a distinct receptor tyrosine kinase known elk.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of Ser. No. 08/240,124,filed May 9, 1994, now U.S. Pat. No. 5,516,658 is a continuation-in-partof application Ser. No. 08/161,132, filed Dec. 3, 1993, now abandoned,which is a continuation-in-part of application Ser. No. 08/114,426,filed Aug. 30, 1993, now abandoned, which is a continuation-in-part ofapplication Ser. No. 08/109,745, filed Aug. 20, 1993, now abandoned.

BACKGROUND OF THE INVENTION

Proteins known as the receptor tyrosine kinases have an intrinsic kinaseactivity that is activated upon ligand binding. This class of proteinsis characterized by conserved structural motifs within the catalyticdomains (Hanks et al., Science, 242:42, 1988) and can be subdivided intofamilies based on structural features of the regions 5' to the catalyticdomain.

Boyd et al. (J. Biol. Chem., 267:3262, 1992) purified a cell surfaceglycoprotein exhibiting tyrosine kinase activity. The N-terminal aminoacid sequence identified this protein as a member of the eph/elk family,and the protein was thus designated hek (human eph/elk-like kinase). Amonoclonal antibody immunoreactive with hek was used to study hekexpression on a number of human cell types (Boyd et al., supra). Hekantigen was detected on the human pre-B cell leukemia cell line LK63(the cell line employed as the immunogen against which the antibody wasraised) and the human T-cell leukemia cell line JM. The Raji B lymphomacell line showed weak hek antigen expression, and the remaining celllines tested (both normal and tumor cell lines, among which werehemopoietic cell lines that included pre-B and T-cell lines) wereconsistently negative. Of the normal and tumor tissue biopsy specimensthat were also tested for hek antigen expression, none of the normaltissues was positive and only a very low proportion of hemopoietictumors was positive.

Expression of hek transcripts on the above-described LK63 and JM celllines, as well as on the human T-cell leukemia cell line HSB-2, has beendemonstrated by northern blot analysis (Wicks et al., Proc. Natl. Acad.Sci. USA, 89:1611, 1992). Nucleotide and amino acid sequences for anisolated hek cDNA clone are presented in Wicks et al., supra.

The hek protein is very closely related to a number of other receptortyrosine kinases, including elk (Letwin et al., Oncogene 3:621, 1988 andLhotak et al., Mol. Cell. Biol. 11:2496, 1991); the hek homologs mek4and cek4 (Sajjadi et al. New Biol. 3:769, 1991); eek (Chan et al.Oncogene 6:1057, 1991); erk (Chan et al. supra.), eck (Lindberg et al.Mol. Cell. Biol. 10:6316, 1990); cek5 (Pasquale, E. B. Cell Regulation2:523, 1991); and eph (Hirai et al. Science 238:1717, 1987). Theproteins of this subfamily are related not only in their cytoplasmicdomains, but also in their extracellular domains, which are 41 to 68%identical. Interestingly, the tissue distributions of these variousreceptors are diverse. For example, expression of elk mRNA has beenreported to be limited to testis and brain (Lhotak et al., supra),whereas eck is found not only in these same two tissues but in lung,intestine, kidney, spleen, ovary, and skin as well.

Ligands for the receptor tyrosine kinases are a diverse group ofproteins that affect the growth, differentiation, and survival of cellsexpressing the receptors. To date, no ligand for hek has beendiscovered. Identification of the putative ligand or ligands that bindhek would prove useful in investigating the nature of cellular processesregulated by the hek protein.

SUMMARY OF THE INVENTION

The present invention provides novel cytokines designated hek ligands(hek-L) that bind to the cell surface receptor known as hek. The presentinvention also provides isolated DNA encoding the hek-L proteins,expression vectors comprising the isolated DNA, and a method forproducing hek-L by cultivating host cells containing the expressionvectors under conditions appropriate for expression of the hek-Lprotein. Antibodies directed against hek-L proteins or an immunogenicfragment thereof are also disclosed.

DETAILED DESCRIPTION OF THE INVENTION

cDNAs encoding novel protein ligands that bind to the cell surfaceprotein known as hek have been isolated in accordance with the presentinvention. Also provided are expression vectors comprising the hekligand (hek-L) cDNA and methods for producing recombinant hek-Lpolypeptides by cultivating host cells containing the expression vectorsunder conditions appropriate for expression of hek-L, and recovering theexpressed hek-L. Purified hek-L protein is also encompassed by thepresent invention, including soluble forms of the protein.

The present invention also provides hek-L or antigenic fragments thereofthat can act as immunogens to generate antibodies specific to the hek-Limmunogens. Monoclonal antibodies specific for hek-L or antigenicfragments thereof thus can be prepared.

The novel cytokines disclosed herein are ligands for hek, a cell surfacereceptor that is a member of the receptor tyrosine kinase family. Oneuse of the hek ligands of the present invention is as research tools forstudying the role that hek-L, in conjunction with hek, may play ingrowth or differentiation of cells bearing the hek receptor. Biologicalsignals that may be initiated by binding of a hek-L to hek on a cell canbe investigated. The possibility that hek plays a role in tumorigenesishas been suggested (Boyd et al., supra). The hek ligands provided hereinare useful for studying what effect binding of hek-L to the cognatereceptor may have on tumorigenesis.

The hek-L polypeptides of the present invention also may be employed inin vitro assays for detection of hek or hek-L or the interactionsthereof. Since hek antigen has been detected on certain leukemic celllines, hek-L may be employed as a carrier to deliver diagnostic orcytotoxic agents to such cells. These and other uses of hek ligands arefurther discussed below.

The hek-L proteins of the present invention also have been found to bindto the receptor tyrosine kinase known as elk. Elk has been described byLetwin et al., Oncogene 3:621, 1988 and Lhotak et al., Mol. Cell. Biol.11:2496, 1991. Scatchard analysis revealed a biphasic pattern of elkbinding for both hek-L proteins, as described in Example 5. Thus, thehek-L proteins disclosed herein also may be employed to bind elk, e.g.,in various assay procedures. However, the elk ligand (elk-L) proteindescribed in Example 5 generally would be preferred for such uses inview of the higher affinity of elk-L for elk.

The binding studies described in Example 5 also revealed that elk ligand(elk-L) binds hek (biphasic binding pattern). A related protein known asB61 (Holzman et al., Mol. Cell. Biol. 10:5830, 1990) was found to bindboth hek (linear pattern) and elk (biphasic pattern). The relativeaffinities are shown in Tables I and II of Example 5.

To identify cells suitable for use as nucleic acid sources in theattempt to clone hek-L DNA, different types of cells were screened forthe ability to bind hek (in the form of a fusion protein comprisinghuman hek and an antibody Fc polypeptide). A human T-cell leukemia cellline was positive for hek/Fc binding, and a cDNA expression library wasderived therefrom. Two distinct cDNA clones encoding human hek-L weresuccessfully isolated by screening clones for expression of ahek/Fc-binding protein, as described in Example 3. The DNA sequence andencoded amino acid sequence of one human hek-L cDNA clone are set forthin SEQ ID NO:1 and SEQ ID NO:2. DNA and encoded amino acid sequences ofa second human hek-L clone are presented in SEQ ID NO:3 and SEQ ID NO:4.Comparison of both the nucleotide and encoded amino acid sequences ofthe human hek-L cDNA clones with the Genbank and Swisspro databasesshowed that the sequences of the hek ligands were unique. The amino acidsequences of the hek-binding proteins encoded by the two clones are 38%identical.

Human hek-L cDNA was isolated from the first positive clone and insertedinto the Bam HI site (in the multiple cloning site region) of cloningvector pBLUESCRIPT® SK(-), available from Stratagene Cloning Systems, LaJolla, Calif. The resulting recombinant vector, designated A2/pBS, in E.coli DH5α cells, was deposited with the American Type Culture Collectionon Aug. 11, 1993, and assigned accession no. ATCC 69384. Human hek-LcDNA was isolated from the second positive clone and inserted into theBam HI site of pBLUESCRIPT® SK(-). The resulting recombinant vector,designated C6/pBS, in E. coli DH5α cells, was deposited with theAmerican Type Culture Collection on Aug. 25, 1993, and assignedaccession no. ATCC 69395. Both deposits were made under the terms of theBudapest Treaty.

The hek-L of SEQ ID NO:2 (encoded by the cDNA of clone A2) comprises anN-terminal signal peptide (amino acids -19 through -1), an extracellulardomain (amino acids 1 through 202), and a C-terminal hydrophobic regionthat begins with amino acid 203. The hek-L of SEQ ID NO:4 (encoded bythe cDNA of clone C6) comprises an N-terminal terminal signal peptide(amino acids -22 through -1), an extracellular domain (amino acids 1through 160), and a C-terminal hydrophobic region that begins with aminoacid 161.

The hek-L proteins expressed by clones A2 and C6 were found to beanchored to the cell surface via glycosyl-phosphatidylinositol (GPI)linkage. GPI membrane anchors, including the chemical structure andprocessing thereof, are described in Ferguson, M. and A. Williams, Ann.Rev. Biochem., 57:285, 1988 (hereby incorporated by reference). Wheninitially expressed, certain proteins comprise a C-terminal hydrophobicdomain that contains signals for GPI anchoring. A cleavage site islocated upstream, often about 10-12 amino acids upstream of theN-terminus of the hydrophobic domain. Post-translational processingincludes cleavage of the protein at this cleavage site. A GPI anchorattaches to the newly exposed C-terminal amino acid of the processed,mature protein. Thus, when the hek-L proteins are expressed in cellsthat recognize the GPI anchoring signals in the hydrophobic domain, thefull length amino acid sequences of SEQ ID NOS:2 and 4 representprecursor forms of the proteins.

Based on consensus sequences derived from other GPI-anchored proteins,likely cleavage sites in the hek-L proteins of the present invention arebetween amino acids 194 and 195 of SEQ ID NO:2 and between amino acids148 and 149 of SEQ ID NO:4. After cleavage of the protein, a GPI moietyattaches to the serine residue that is now the C-terminus of theprocessed protein (amino acid 194 of SEQ ID NO:2 and amino acid 148 ofSEQ ID NO:4). It is possible that cleavage occurs elsewhere upstream ofthe hydrophobic region in the hek-L proteins.

The term "hek-L" as used herein refers to a genus of polypeptides whichare capable of binding hek and exhibit homology (preferably being atleast 80% homologous) to the hek-L protein of SEQ ID NO:2 or SEQ IDNO:4. Human hek-L is within the scope of the present invention, as arehek-L proteins derived from other mammalian species including but notlimited to murine, rat, bovine, porcine, or various primate species. Asused herein, the term "hek-L" includes both membrane-bound and soluble(secreted) forms of the protein. Truncated proteins that retain thehek-binding property are encompassed by the present invention. Suchtruncated proteins include, for example, soluble hek-L comprising onlythe extracellular (receptor binding) domain but lacking the hydrophobicdomain.

The human hek-L cDNA may be radiolabeled and used as a probe to isolateother mammalian hek-L cDNAs by cross-species hybridization. For example,a cDNA library prepared from T-cell leukemic cell lines of othermammalian species may be screened with radiolabeled human hek-L cDNA toisolate a positive clone. Alternatively, mRNAs isolated from variouscell lines can be screened by Northern hybridization to determine asuitable source of mammalian hek-L mRNA for use in cloning a hek-L gene.

Although a hek/Fc fusion protein was employed in the screeningprocedures described in Examples 2 and 3 below, hek can be used toscreen clones and candidate cell lines for expression of hek-L proteins.The hek/Fc fusion protein, however, offers the advantage of being easilypurified. In addition, disulfide bonds form between the Fc regions oftwo separate fusion protein chains, creating dimers.

Other antibody Fc regions may be substituted for the human IgG1 Fcregion mutein described in Example 1. Other suitable Fc regions arethose that can bind with high affinity to protein A or protein G, andinclude the Fc region of murine IgG1 or fragments of the human IgG1 Fcregion, e.g., fragments comprising at least the hinge region so thatinterchain disulfide bonds will form.

One embodiment of the present invention provides soluble hek-Lpolypeptides. Soluble hek-L polypeptides comprise all or part of theextracellular domain of a native hek-L but lack the hydrophobic regionthat contains signals that would cause retention of the polypeptide on acell membrane. Soluble hek-L polypeptides advantageously comprise thenative (or a heterologous) signal peptide when initially synthesized topromote secretion, but the signal peptide is cleaved upon secretion ofhek-L from the cell. The soluble hek-L polypeptides that may be employedretain the ability to bind the hek receptor. Soluble hek-L may alsoinclude part of the hydrophobic region provided that the soluble hek-Lprotein is capable of being secreted.

Soluble hek-L may be identified (and distinguished from its non-solublemembrane-bound counterparts) by separating intact cells which expressthe desired protein from the culture medium, e.g., by centrifugation,and assaying the medium (supernatant) for the presence of the desiredprotein. The presence of hek-L in the medium indicates that the proteinwas secreted from the cells and thus is a soluble form of the desiredprotein. Soluble hek-L may be a naturally-occurring form of thisprotein, e.g., arising from alternative splicing. Further, GPI-linkedhek-L may be released or shed from the cell surface into the culturemedium, e.g., by the action of a protease or other enzyme.

The use of soluble forms of hek-L is advantageous for certainapplications. Purification of the proteins from recombinant host cellsis facilitated, since the soluble proteins are secreted from the cells.Further, soluble proteins are generally more suitable for intravenousadministration.

Examples of soluble hek-L polypeptides include those comprising theentire extracellular domain of a native hek-L protein. One such solublehek-L protein comprises amino acids 1 through 202 of SEQ ID NO:2, andanother comprises amino acids 1 through 160 of SEQ ID NO:4. Wheninitially expressed within a host cell, the soluble protein mayadditionally comprise one of the heterologous signal peptides describedbelow that is functional within the host cells employed. Alternatively,the protein may initially comprise the native signal peptide, such thatthe hek-L comprises amino acids -19 through 202 of SEQ ID NO:2 or aminoacids -22 through 160 of SEQ ID NO:4. Soluble hek-L proteins may betruncated to delete the C-terminus up to and including the amino acidthat serves as a GPI attachment site. Examples include proteinscomprising amino acids 1-193 of SEQ ID NO:2 or amino acids 1-147 of SEQID NO:4, as discussed above. Although the GPI attachment site may bedeleted, deletion of the hydrophobic domain is believed to be sufficientto prevent GPI anchoring of the protein to the cell membrane. In furtherembodiments, the proteins may be truncated at the C-terminus so that theC-terminal amino acid is any amino acid between amino acids 193 and 202of SEQ ID NO:2, or between amino acids 147 and 160 of SEQ ID NO:4. DNAsequences encoding soluble hek-L proteins are encompassed by the presentinvention.

Truncated hek-L, including soluble polypeptides, may be prepared by anyof a number of conventional techniques. A desired DNA sequence may bechemically synthesized using known techniques. DNA fragments also may beproduced by restriction endonuclease digestion of a full length clonedDNA sequence, and isolated by electrophoresis on agarose gels.Oligonucleotides that reconstruct the 5' or 3'-terminus of a DNAfragment to a desired point may be synthesized. The oligonucleotide maycontain a restriction endonuclease cleavage site upstream of the desiredcoding sequence and position an initiation codon (ATG) at the 5' end ofthe coding sequence. The well known polymerase chain reaction procedurealso may be employed to isolate a DNA sequence encoding a desiredprotein fragment. As a further alternative, known mutagenesis techniquesmay be employed to insert a stop codon at a desired point, e.g.,immediately downstream of the codon for the last amino acid of theextracellular domain.

Certain embodiments of the present invention provide isolated DNAcomprising a nucleotide sequence selected from the group consisting ofnucleotides 83-796 (entire coding region), 83-745 (encoding the signalpeptide and extracellular domain), 140-796 (encoding the protein withoutthe signal peptide) and 140-745 (encoding the extracellular domain) ofSEQ ID NO:1. Also provided is isolated DNA comprising a nucleotidesequence selected from the group consisting of nucleotides 28-630(entire coding region), 28-573 (encoding the signal peptide andextracellular domain), 94-630 (encoding the protein without the signalpeptide), and 94-573 (encoding the extracellular domain) of SEQ ID NO:3.DNAs encoding biologically active fragments of the proteins of SEQ IDNO:2 and SEQ ID NO:4 are also provided, including but not limited to DNAencoding the above-described hek-L proteins truncated at the C-terminus.

The hek-L DNA of the present invention includes cDNA, chemicallysynthesized DNA, DNA isolated by PCR, genomic DNA, and combinationsthereof. Genomic hek-L DNA may be isolated by hybridization to the cDNAof clones A2 or C6, using standard techniques.

The present invention provides purified hek-L polypeptides, bothrecombinant and non-recombinant. Variants and derivatives of nativehek-L proteins that retain the desired biological activity (e.g., theability to bind hek) are also within the scope of the present invention.In one embodiment of the present invention, mature hek-L protein ischaracterized by the N-terminal amino acid sequenceLeu-Leu-Ala-Gln-Gly-Pro-Gly-Gly-Ala-Leu-Gly-Asn. In another embodiment,mature hek-L protein is characterized by the N-terminal amino acidsequence Gly-Ser-Ser-Leu-Arg-His-Val-Val-Tyr-Trp-Asn-Ser.

hek-L variants may be obtained by mutations of nucleotide sequencescoding for native hek-L polypeptides, for example. A hek-L variant, asreferred to herein, is a polypeptide substantially homologous to anative hek-L, but which has an amino acid sequence different from thatof a native hek-L (human, murine or other mammalian species) because ofone or more deletions, insertions or substitutions. Such variants thatbind hek are equivalents of the native hek-binding proteins having theamino acid sequences presented in SEQ ID NO:2 and SEQ ID NO:4.

Variant DNA and amino acid sequences of the present invention preferablyare at least 80% identical, most preferably at least 90% identical, to anative hek-L sequence such as the native sequences of SEQ ID NOS: 1-4.For fragments, the percent identity is calculated for that portion of anative sequence that is present in the fragment. Certain embodiments ofthe present invention provide hek-L polypeptides comprising an aminoacid sequence that is at least 80% identical to a sequence selected fromthe group consisting of amino acids 1-194, 1-202 and 1-219 of SEQ IDNO:2 and amino acids 1-148, 1-160, and 1-179 of SEQ ID NO:4.

Alterations of the native amino acid sequence may be accomplished by anyof a number of known techniques. Mutations can be introduced atparticular loci by synthesizing oligonucleotides containing a mutantsequence, flanked by restriction sites enabling ligation to fragments ofthe native sequence. Following ligation, the resulting reconstructedsequence encodes an analog having the desired amino acid insertion,substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981);Kunkel (Proc. Natl. Acad. Sci. USA 82:488, 1985); Kunkel et al. (Methodsin Enzymol. 154:367, 1987); and U.S. Pat. Nos. 4,518,584 and 4,737,462,which are incorporated by reference herein.

Variants may comprise conservatively substituted sequences, meaning thata given amino acid residue is replaced by a residue having similarphysiochemical characteristics. Examples of conservative substitutionsinclude substitution of one aliphatic residue for another, such as Ile,Val, Leu, or Ala for one another, or substitutions of one polar residuefor another, such as between Lys and Arg; Glu and Asp; or Gln and Asn.Other such conservative substitutions, for example, substitutions ofentire regions having similar hydrophobicity characteristics, are wellknown.

hek-L also may be modified to create hek-L derivatives by formingcovalent or aggregative conjugates with other chemical moieties, such asglycosyl groups, lipids, phosphate, acetyl groups and the like. Covalentderivatives of hek-L may be prepared by linking the chemical moieties tofunctional groups on hek-L amino acid side chains or at the N-terminusor C-terminus of a hek-L polypeptide or the extracellular domainthereof. Other derivatives of hek-L within the scope of this inventioninclude covalent or aggregative conjugates of hek-L or its fragmentswith other proteins or polypeptides, such as by synthesis in recombinantculture as N-terminal or C-terminal fusions.

hek ligand when initially expressed in a recombinant system may comprisea signal or leader sequence (native or heterologous) at the N-terminusof a hek-L polypeptide. The signal or leader peptide co-translationallyor post-translationally directs transfer of the protein from its site ofsynthesis to a site outside of the cell membrane or cell wall, and iscleaved from the mature protein during the secretion process. Examplesof suitable heterologous signal peptides, which are generally chosenaccording to the expression system to be employed, are described below.

hek-L polypeptide fusions can comprise peptides added to facilitatepurification and identification of hek-L. Such peptides include, forexample, poly-His or the antigenic identification peptides described inU.S. Pat. No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988.One such peptide is the FLAG® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys(DYKDDDDK), which is highly antigenic and provides an epitope reversiblybound by a specific monoclonal antibody, enabling rapid assay and facilepurification of expressed recombinant protein. Fusion proteins cappedwith this peptide may also be resistant to intracellular degradation inE. coli. A murine hybridoma designated 4E11 produces a monoclonalantibody that binds the peptide DYKDDDDK in the presence of certaindivalent metal cations (as described in U.S. Pat. No. 5,011,912, herebyincorporated by reference) and has been deposited with the American TypeCulture Collection under accession no. HB 9259.

The present invention further includes hek-L polypeptides with orwithout associated native-pattern glycosylation. hek-L expressed inyeast or mammalian expression systems (e.g., COS-7 cells) may be similarto or significantly different from a native hek-L polypeptide inmolecular weight and glycosylation pattern, depending upon the choice ofexpression system. Expression of hek-L polypeptides in bacterialexpression systems, such as E. coli, provides non-glycosylatedmolecules.

DNA constructs that encode various additions or substitutions of aminoacid residues or sequences, or deletions of terminal or internalresidues or sequences not needed for biological activity or binding canbe prepared. For example, N-glycosylation sites in the hek-Lextracellular domain can be modified to preclude glycosylation, allowingexpression of a more homogeneous, reduced carbohydrate analog inmammalian and yeast expression systems. N-glycosylation sites ineukaryotic polypeptides are characterized by an amino acid tripletAsn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr.Appropriate modifications to the nucleotide sequence encoding thistriplet will result in substitutions, additions or deletions thatprevent attachment of carbohydrate residues at the Asn side chain.Alteration of a single nucleotide, chosen so that Asn is replaced by adifferent amino acid, for example, is sufficient to inactivate anN-glycosylation site. Known procedures for inactivating N-glycosylationsites in proteins include those described in U.S. Pat. No. 5,071,972 andEP 276,846, hereby incorporated by reference.

Three N-glycosylation sites are found in the hek-L encoded by clone A2,at amino acids 19-21, 48-50, and 81-83 of SEQ ID NO:2. OneN-glycosylation site is found in the hek-L encoded by clone C6, at aminoacids 11-13 of SEQ ID NO:4.

In another example, sequences encoding Cys residues that are notessential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges uponrenaturation. Other variants are prepared by modification of adjacentdibasic amino acid residues to enhance expression in yeast systems inwhich KEX2 protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites. KEX2protease processing sites are found in the hek-L of SEQ ID NO:2 at aminoacids 26-27, 87-88, and 199-200. The hek-L of SEQ ID NO:4 comprises KEX2protease processing sites at amino acids 73-74 and 134-135.

Naturally occurring hek-L variants are also encompassed by the presentinvention. Examples of such variants are proteins that result fromalternative mRNA splicing events or from proteolytic cleavage of thehek-L protein, wherein the hek-binding property is retained. Alternativesplicing of mRNA may yield a truncated but biologically active hek-Lprotein, such as a naturally occurring soluble form of the protein, forexample. Variations attributable to proteolysis include, for example,differences in the N- or C-termini upon expression in different types ofhost cells, due to proteolytic removal of one or more terminal aminoacids from the hek-L protein (generally from 1-5 terminal amino acids).Signal peptides may be cleaved at different positions in a givenprotein, resulting in variations of the N-terminal amino acid of themature protein.

In one expression system, the N-terminal amino acid of a hek-L proteinencoded by clone C6 was amino acid 4 (Leu) of SEQ ID NO:4. Onepreparation of a soluble hek-L/Fc fusion protein derived from clone A2comprised a mixture of fusion proteins having amino acid 12 (Asn) of SEQID NO:2 as the N-terminal amino acid (about 60%) and fusion proteins inwhich amino acid 1 (Leu) of SEQ ID NO:2 was the N-terminal amino acid.Certain embodiments of the present invention thus are directed toproteins (soluble or membrane-bound) in which the N-terminal amino acidis any of amino acids 1-4 of SEQ ID NO:4, or any of amino acids 1-12 ofSEQ ID NO:2.

Due to the known degeneracy of the genetic code wherein more than onecodon can encode the same amino acid, a DNA sequence may vary from thatpresented in SEQ ID NOS:1 or 3, and still encode a hek-L protein havingthe amino acid sequence of SEQ ID NOS:2 or 4. Such variant DNA sequencesmay result from silent mutations (e.g., occurring during PCRamplification), and may be the product of deliberate mutagenesis of anative sequence.

The present invention thus provides isolated DNA sequences encodingbiologically active hek-L, selected from: (a) DNA derived from thecoding region of a native mammalian hek-L gene (e.g., cDNA comprisingthe coding region of the nucleotide sequence presented in SEQ ID NO:1 orSEQ ID NO:3); and (b) DNA which is degenerate as a result of the geneticcode to a DNA defined in (a) and which encodes biologically activehek-L. The hek-L proteins encoded by such DNA sequences are encompassedby the present invention.

The hek-L DNA of the present invention includes cDNA, chemicallysynthesized DNA, DNA isolated by PCR, genomic DNA, and combinationsthereof. Gemomic hek-L DNA may be isolated by hybridization to the cDNAof clones A2 or C6, using standard techniques.

Assays for Biological Activity

Variants possessing the ability to bind hek may be identified by anysuitable assay. Conventional assay techniques also are useful foranalyzing the hek-binding activity of a native hek-L protein. Biologicalactivity of hek-L may be determined, for example, by competition forbinding to the ligand binding domain of hek (i.e. competitive bindingassays).

One type of a competitive binding assay for hek-L polypeptide uses aradiolabeled, soluble human hek-L and intact cells expressing cellsurface hek. Instead of intact cells, one could substitute soluble hek(such as a hek/Fc fusion protein) bound to a solid phase through aProtein A or Protein G interaction with the Fc region of the fusionprotein. Another type of competitive binding assay utilizes radiolabeledsoluble hek such as a hek/Fc fusion protein, and intact cells expressinghek-L. Alternatively, soluble hek-L could be bound to a solid phase.

Competitive binding assays can be performed using standard methodology.For example, radiolabeled hek-L can be used to compete with a putativehek-L homolog to assay for binding activity against surface-bound hek.Qualitative results can be obtained by competitive autoradiogaphic platebinding assays, or Scatchard plots may be utilized to generatequantitative results.

Alternatively, soluble hek can be bound to a solid phase such as acolumn chromatography matrix or a similar substrate suitable foranalysis for the presence of a detectable moiety such as ¹²⁵ I-labeledhek-L. Binding to a solid phase can be accomplished, for example, bybinding a hek/Fc fusion protein to a protein A or protein G-containingmatrix.

The binding characteristics of hek-L (including variants) may also bedetermined using labeled, soluble hek (for example, ¹²⁵ I-hek/Fc) incompetition assays similar to those described above. In this case,however, intact cells expressing hek-L, or soluble hek-L bound to asolid substrate, are used to measure the extent to which a samplecontaining a putative hek variant competes for binding of a labeledsoluble hek to hek-L.

A preferred assay for detecting hek-binding activity of a membrane-boundhek-L is as follows. A modified indirect binding assay was devised,using the hek/Fc fusion protein prepared in Example 1 and the ¹²⁵I-labeled mouse anti-human IgG Fc antibody described in Example 3 toavoid direct radiolabeling of hek/Fc. Cells expressing endogenous hek-L(e.g., the CCRF-HSB-2 cell line described in Example 2) are exposed tovarying concentrations of hek/Fc, followed by a constant saturatingconcentration of the ¹²⁵ I-antibody as follows.

CCRF-HSB-2 cells are cultivated in suspension culture in 96-well cultureplates. The cells (2×10⁶ cells/well) are incubated in the presence orabsence of various concentrations of hek/Fc in binding medium (RPMI 1640medium, 1% bovine serum albumin, 0.2% sodium azide and 20 mM Hepes, pH7.2) for one hour at 37° C. Cells are then washed once with PBS andincubated with ¹²⁵ I-mouse anti-human IgG Fc (40 ng/ml) in bindingmedium with gentle agitation for one hour at 37° C. Cells and unbound¹²⁵ I-antibody are separated by the pthalate oil separation method,essentially as described by Dower et al., J. Immunol. 132:751 (1984).

An assay for hek/Fc-binding to cells expressing recombinantmembrane-bound hek-L may be conducted as described in Example 5. Anindirect binding assay was employed.

A preferred assay for analyzing the hek-binding activity of solublehek-L is as follows. The assay detects the ability of a soluble hek-L toinhibit binding of a hek/Fc fusion protein to the CCRF-HSB-2 cell linethat expresses endogenous hek-L, as described in Example 2.

Conditioned supernatant (culture medium) from CV-1/EBNA cellstransfected with an expression vector expressing a soluble hek-L istitrated in a 96-well plate. A constant amount of hek/Fc (1 μg/well) isadded to each well, followed by 1-2×10⁶ CCRF-HSB-2 cells per well, inbinding medium. The plate is incubated at 37° C. for one hour. Cells arewashed twice with PBS, then pelleted by centrifugation. ¹²⁵ I-mouseanti-human IgG Fc is added to each well at a constant concentration, andthe plate is incubated for an additional hour at 37° C. The ¹²⁵ I-mouseanti-human IgG Fc binds to the hek/Fc that bound to the CCRF-HSB-2cells. After the final incubation, cells are harvested over phthalateoil-containing tubes to separate the bound and free ¹²⁵ I-mouseanti-human IgG Fc. The radioactivity is quantitated using a gammacounter.

Uses of hek-L

The hek-L of the present invention can be used in a binding assay todetect cells expressing hek. For example, hek-L or the extracellulardomain or a fragment thereof can be conjugated to a detectable moietysuch as ¹²⁵ I. Radiolabeling with ¹²⁵ I can be performed by any ofseveral standard methodologies that yield a functional ¹²⁵ I-hek-Lmolecule labeled to high specific activity. Alternatively, anotherdetectable moiety such as an enzyme that can catalyze a colorometric orfluorometric reaction, biotin or avidin may be used. Cells to be testedfor hek expression can be contacted with labeled hek-L. Afterincubation, unbound labeled hek-L is removed and binding is measuredusing the detectable moiety.

The hek ligand proteins disclosed herein also may be employed to measurethe biological activity of hek protein in terms of binding affinity forhek-L. To illustrate, hek-L may be employed in a binding affinity studyto measure the biological activity of a hek protein that has been storedat different temperatures, or produced in different cell types. Thebiological activity of a hek protein thus can be ascertained before itis used in a research study, for example.

Hek-L proteins find use as reagents that may be employed by thoseconducting "quality assurance" studies, e.g., to monitor shelf life andstability of hek protein under different conditions. Hek ligands may beused in determining whether biological activity is retained aftermodification of a hek protein (e.g., chemical modification, truncation,mutation, etc.). The binding affinity of the modified hek protein for ahek-L is compared to that of an unmodified hek protein to detect anyadverse impact of the modification on biological activity of hek.

A different use of a hek ligand is as a reagent in protein purificationprocedures. Hek-L or hek-L/Fc fusion proteins may be attached to a solidsupport material by conventional techniques and used to purify hek byaffinity chromatography.

Hek-L polypeptides also find use as carriers for delivering agentsattached thereto to cells bearing the hek cell surface antigen.Expression of hek antigen has been reported for certain leukemic celllines, including the human T-cell leukemia cell lines designated JM andHSB-2 and the human pre-B cell leukemia cell line designated LK63 (Boydet al., J. Biol. Chem. 267:3262, 1992, and Wicks et al., Proc. Nat.Acad. Sci. USA, 89:1611, 1992). Hek-L proteins thus can be used todeliver diagnostic or therapeutic agents to these cells (or to othercell types found to express hek on the cell surface) in in vitro or invivo procedures.

One example of such use is to expose a hek⁺ leukemic cell line to atherapeutic agent/hek-L conjugate to assess whether the agent exhibitscytotoxicity toward the leukemia cells. A number of differenttherapeutic agents attached to hek-L may be included in an assay todetect and compare the cytotoxic effect of the agents on the leukemiacells. Hek-L/diagnostic agent conjugates may be employed to detect thepresence of hek⁺ cells in vitro or in vivo.

Diagnostic and therapeutic agents that may be attached to a hek-Lpolypeptide include, but are not limited to, drugs, toxins,radionuclides, chromophores, fluorescent compounds, enzymes thatcatalyze a colorimetric or fluorometric reaction, and the like, with theparticular agent being chosen according to the intended application.Examples of drugs include those used in treating various forms ofcancer, e.g., nitrogen mustards such as L-phenylalanine nitrogen mustardor cyclophosphamide, intercalating agents such ascis-diaminodichloroplatinum, antimetabolites such as 5-fluorouracil,vinca alkaloids such as vincristine, and antibiotics such as bleomycin,doxorubicin, daunorubicin, and derivatives thereof. Among the toxins arericin, abrin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A,ribosomal inactivating proteins, mycotoxins such as trichothecenes, andderivatives and fragments (e.g., single chains) thereof. Radionuclidessuitable for diagnostic use include, but are not limited to, ¹²³ I, ¹³¹I, ^(99m) Tc, ¹¹¹ In, and ⁷⁶ Br. Radionuclides suitable for therapeuticuse include, but are not limited to, ¹³¹ I, ²¹¹ At, ⁷⁷ Br, ¹⁸⁶ Re, ¹⁸⁸Re, ²¹² Pb, ²¹² Bi, ¹⁰⁹ Pd, ⁶⁴ Cu, and ⁶⁷ Cu.

Such agents may be attached to the hek-L by any suitable conventionalprocedure. Hek-L, being a protein, comprises functional groups on aminoacid side chains that can be reacted with functional groups on a desiredagent to form covalent bonds, for example. Alternatively, the protein oragent may be derivatized to generate or attach a desired reactivefunctional group. The derivatization may involve attachment of one ofthe bifunctional coupling reagents available for attaching variousmolecules to proteins (Pierce Chemical Company, Rockford, Ill.). Anumber of techniques for radiolabeling proteins are known. Radionuclidemetals may be attached to hek-L by using a suitable bifunctionalchelating agent, for example.

Conjugates comprising hek-L and a suitable diagnostic or therapeuticagent (preferably covalently linked) are thus prepared. The conjugatesare administered or otherwise employed in an amount appropriate for theparticular application.

As described in Example 5, the hek-L proteins provided herein also arecapable of binding a receptor known as elk. Thus, hek-L has additionaluses stemming from the elk-binding property, analogous to those usesdescribed above that stemmed from the hek-binding property. Hek-L can beused to detect elk in various assays. An antibody that binds hek may beemployed in an assay, if appropriate, to block binding of hek to hek-Lwhile allowing binding of elk to hek-L. Hek ligands may be employed inassessing the biological activity of elk proteins in terms of thebinding affinity of an elk protein or variant thereof for a hek-L. Hek-Lproteins also find use in purifying elk proteins by affinitychromatography.

Hek-L Oligomers

The present invention encompasses hek-L polypeptides in the form ofoligomers, such as dimers or trimers. Oligomers may be formed bydisulfide bonds between cysteine residues on different hek-Lpolypeptides. In one embodiment of the invention, a hek-L dimer iscreated by fusing hek-L to the Fc region of an antibody (IgG1) in amanner that does not interfere with binding of hek-L to the hek ligandbinding domain. The term "Fc polypeptide" includes native and muteinforms, as well as truncated Fc polypeptides containing the hinge regionthat promotes dimerization. The Fc polypeptide preferably is fused tothe C-terminus of a soluble hek-L (comprising only the extracellulardomain). Preparation of fusion proteins comprising heterologouspolypeptides fused to various portions of antibody-derived polypeptides(including the Fc domain) has been described, e.g., by Ashkenazi et al.(PNAS USA 88:10535, 1991) and Byrn et al. (Nature 344:677, 1990), herebyincorporated by reference.

A fusion of the hek-L to an Fc or Fc mutein polypeptide may be preparedby procedures analogous to those described in Example 1 for preparationof a hek/Fc mutein fusion. A gene fusion encoding the hek-L/Fc fusionprotein is inserted into an appropriate expression vector andtransfected into host cells. The expressed hek-L/Fc fusion proteins areallowed to assemble much like antibody molecules, whereupon interchaindisulfide bonds form between Fc polypeptides, yielding divalent hek-L.Alternatively, the native Fc polypeptide from which the mutein wasderived may be employed.

If fusion proteins are made with both heavy and light chains of anantibody, it is possible to form a hek-L oligomer with as many as fourhek-L extracellular regions. Alternatively, one can link two solublehek-L domains with a peptide linker such as those described in U.S. Pat.No. 5,073,627.

In particular embodiments of the present invention, hek-L DNA encodingamino acids -19 through 202 of SEQ ID NO:1 or amino acids -22 through157 of SEQ ID NO:3 was fused to the 5' end of DNA encoding the Fc muteindescribed in Example 1, and inserted into the expression vector pDC410(described in Example 3). CV1-EBNA-1 cells transfected with theresulting recombinant expression vector were cultivated to express thesoluble hek-L/Fc fusion protein.

The present invention provides oligomers of hek-L extracellular domainsor fragments thereof, linked by disulfide interactions, or expressed asfusion polymers with or without spacer amino acid linking groups. Forexample, a dimer of the hek-L extracellular domain can be linked by anIgG Fc region linking group.

Expression Systems

The present invention provides recombinant expression vectors forexpression of hek-L, and host cells transformed with the expressionvectors. Any suitable expression system may be employed. The vectorsinclude a hek-L DNA sequence operably linked to suitable transcriptionalor translational regulatory nucleotide sequences, such as those derivedfrom a mammalian, microbial, viral, or insect gene. Examples ofregulatory sequences include transcriptional promoters, operators, orenhancers, an mRNA ribosomal binding site, and appropriate sequenceswhich control transcription and translation initiation and termination.Nucleotide sequences are operably linked when the regulatory sequencefunctionally relates to the hek-L DNA sequence. Thus, a promoternucleotide sequence is operably linked to a hek-L DNA sequence if thepromoter nucleotide sequence controls the transcription of the hek-L DNAsequence. The ability to replicate in the desired host cells, usuallyconferred by an origin of replication, and a selection gene by whichtransformants are identified, may additionally be incorporated into theexpression vector.

In addition, sequences encoding appropriate signal peptides that are notnative to the hek-L gene can be incorporated into expression vectors.For example, a DNA sequence for a signal peptide (secretory leader) maybe fused in frame to the hek-L sequence so that the hek-L is initiallytranslated as a fusion protein comprising the signal peptide. A signalpeptide that is functional in the intended host cells enhancesextracellular secretion of the hek-L polypeptide. The signal peptide iscleaved from the hek-L polypeptide upon secretion of hek-L from thecell.

Suitable host cells for expression of hek-L polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-freetranslation systems could also be employed to produce hek-L polypeptidesusing RNAs derived from DNA constructs disclosed herein.

Prokaryotes include gram negative or gram positive organisms, forexample, E. coli or Bacilli. Suitable prokaryotic host cells fortransformation include, for example, E. coli, Bacillus subtilis,Salmonella typhimurium, and various other species within the generaPseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic hostcell, such as E. coli, a hek-L polypeptide may include an N-terminalmethionine residue to facilitate expression of the recombinantpolypeptide in the prokaryotic host cell. The N-terminal Met may becleaved from the expressed recombinant hek-L polypeptide.

Expression vectors for use in prokaryotic phenotypic selectable compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. An appropriate promoter and a hek-L DNA sequence areinserted into the pBR322 vector. Other commercially available vectorsinclude, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden) and pGEM 1 (Promega Biotec, Madison, Wis., USA).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include β-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412, 1982). A particularly useful prokaryotic host cell expressionsystem employs a phage λ P_(L) promoter and a cI857ts thermolabilerepressor sequence. Plasmid vectors available from the American TypeCulture Collection which incorporate derivatives of the λ P_(L) promoterinclude plasmid pHUB2 (resident in E. coli strain JMB9 (ATCC 37092)) andpPLc28 (resident in E. coli RR1 (ATCC 53082)).

hek-L alternatively may be expressed in yeast host cells, preferablyfrom the Saccharomyces genus (e.g., S. cerevisiae). Other genera ofyeast, such as Pichia or Kluyveromyces, may also be employed. Yeastvectors will often contain an origin of replication sequence from a 2 μyeast plasmid, an autonomously replicating sequence (ARS), a promoterregion, sequences for polyadenylation, sequences for transcriptiontermination, and a selectable marker gene. Suitable promoter sequencesfor yeast vectors include, among others, promoters for metallothionein,3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073,1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg.7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Other suitable vectors andpromoters for use in yeast expression are further described in Hitzeman,EPA-73,657. Another alternative is the glucose-repressible ADH2 promoterdescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast andE. coli may be constructed by inserting DNA sequences from pBR322 forselection and replication in E. coli (Amp^(r) gene and origin ofreplication) into the above-described yeast vectors.

The yeast α-factor leader sequence may be employed to direct secretionof the hek-L polypeptide. The α-factor leader sequence is often insertedbetween the promoter sequence and the structural gene sequence. See,e.g., Kurjan et al., Cell 30:933, 1982; Bitter et al., Proc. Natl. Acad.Sci. USA 81:5330, 1984; U.S. Pat. No. 4,546,082; and EP 324,274. Otherleader sequences suitable for facilitating secretion of recombinantpolypeptides from yeast hosts are known to those of skill in the art. Aleader sequence may be modified near its 3' end to contain one or morerestriction sites. This will facilitate fusion of the leader sequence tothe structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/ml adenine and 20 μg/ml uracil.

Yeast host cells transformed by vectors containing ADH2 promotersequence may be grown for inducing expression in a "rich" medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian or insect host cell culture systems could also be employed toexpress recombinant hek-L polypeptides. Baculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, Bio/Technology 6:47 (1988). Established cell linesof mammalian origin also may be employed. Examples of suitable mammalianhost cell lines include the COS-7 line of monkey kidney cells (ATCC CRL1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, andBHK (ATCC CRL 10) cell lines, and the CV-1/EBNA-1 cell line derived fromthe African green monkey kidney cell line CV1 (ATCC CCL 70) as describedby McMahan et al. (EMBO J. 10: 2821, 1991).

Transcriptional and translational control sequences for mammalian hostcell expression vectors may be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from Polyomavirus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites may be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment which may also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40fragments may also be used, provided the approximately 250 bp sequenceextending from the HindIII site toward the BglI site located in the SV40viral origin of replication site is included.

Exemplary expression vectors for use in mammalian host cells can beconstructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280,1983). A useful system for stable high level expression of mammaliancDNAs in C 127 murine mammary epithelial cells can be constructedsubstantially as described by Cosman et al. (Mol. Immunol. 23:935,1986). A useful high expression vector, PMLSV N1/N4, described by Cosmanet al., Nature 312:768, 1984 has been deposited as ATCC 39890.Additional useful mammalian expression vectors are described inEP-A-0367566, and in U.S. patent application Ser. No. 07/701,415, filedMay 16, 1991, incorporated by reference herein. The vectors may bederived from retroviruses.

In place of DNA encoding the native signal sequence, the vector maycontain DNA encoding a heterologous signal sequence. Examples includethe signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No.4,965,195; the signal sequence for interleukin-2 receptor described inCosman et al., Nature 312:768 (1984); the interleukin-4 signal peptidedescribed in EP 367,566; the type I interleukin-1 receptor signalpeptide described in U.S. Pat. No. 4,968,607; and the type IIinterleukin-1 receptor signal peptide described in EP 460,846.

Protein Purification

The present invention provides substantially homogeneous hek-L protein,which may be produced by recombinant expression systems as describedabove or purified from naturally occurring cells. The hek-L is purifiedto substantial homogeneity, as indicated by a single protein band uponanalysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

One process for producing the hek-L protein comprises culturing a hostcell transformed with an expression vector comprising a DNA sequencethat encodes hek-L under conditions such that hek-L is expressed. Thehek-L protein is then recovered from culture medium or cell extracts,depending upon the expression system employed. As the skilled artisanwill recognize, procedures for purifying the recombinant hek-L will varyaccording to such factors as the type of host cells employed and whetheror not the hek-L is secreted into the culture medium.

For example, when expression systems that secrete the recombinantprotein are employed, the culture medium first may be concentrated usinga commercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.Finally, one or more reversed-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,(e.g., silica gel having pendant methyl or other aliphatic groups) canbe employed to further purify hek-L. Some or all of the foregoingpurification steps, in various combinations, can be employed to providea substantially homogeneous recombinant protein.

It is also possible to utilize an affinity column comprising the ligandbinding domain of hek to affinity-purify expressed hek-L polypeptides.hek-L polypeptides can be removed from an affinity column in a high saltelution buffer and then dialyzed into a lower salt buffer for use.Alternatively, the affinity column may comprise an antibody that bindshek-L. In a further alternative, an affinity column comprises a hek/Fcfusion protein bound to a Protein A column.

Recombinant protein produced in bacterial culture is usually isolated byinitial disruption of the host cells, centrifugation, extraction fromcell pellets if an insoluble polypeptide, or from the supernatant fluidif a soluble polypeptide, followed by one or more concentration,salting-out, ion exchange, affinity purification or size exclusionchromatography steps. Finally, RP-HPLC can be employed for finalpurification steps. Microbial cells can be disrupted by any convenientmethod, including freeze-thaw cycling, sonication, mechanicaldisruption, or use of cell lysing agents.

Transformed yeast host cells may be employed to express hek-L as asecreted polypeptide. Secreted recombinant polypeptide from a yeast hostcell fermentation can be purified by methods analogous to thosedisclosed by Urdal et al. (J. Chromatog. 296:171, 1984). Urdal et al.describe two sequential, reversed-phase HPLC steps for purification ofrecombinant human IL-2 on a preparative HPLC column.

The present invention provides pharmaceutical compositions comprising ahek-L polypeptide and a physiologically acceptable carrier, diluent, orexcipient. Such compositions may comprise buffers, antioxidants such asascorbic acid, low molecular weight (less than about 10 residues)polypeptides, proteins, amino acids, carbohydrates including glucose,sucrose, or dextrins, chelating agents such as EDTA, glutathione andother stabilizers and excipients. Neutral buffered saline or salinemixed with conspecific serum albumin are exemplary appropriate diluents.

Nucleic Acid Fragments

The present invention further provides fragments of the hek-L nucleotidesequences presented herein. Such fragments desirably comprise at leastabout 14 nucleotides of the sequence presented in SEQ ID NOS:1 or 3. DNAand RNA complements of said fragments are provided herein, along withboth single-stranded and double-stranded forms of the hek-L DNA.

Among the uses of such hek-L nucleic acid fragments is use as a probe.Such probes may be employed in cross-species hybridization procedures toisolate hek-L DNA from additional mammalian species. As one example, aprobe corresponding to the extracellular domain of hek-L may beemployed. The probes also find use in detecting the presence of hek-Lnucleic acids in in vitro assays and in such procedures as Northern andSouthern blots. Cell types expressing hek-L can be identified. Suchprocedures are well known, and the skilled artisan can choose a probe ofsuitable length, depending on the particular intended application.

Other useful fragments of the hek-L nucleic acids are antisense or senseoligonucleotides comprise a single-stranded nucleic acid sequence(either RNA or DNA) capable of binding to target hek-L mRNA (sense) orhek-L DNA (antisense) sequences. Antisense or sense oligonucleotides,according to the present invention, may comprise a fragment of thecoding region of hek-L cDNA shown in SEQ ID NO:1 or SEQ ID NO:3, or theDNA or RNA complement thereof. Such a fragment generally comprises atleast about 14 nucleotides, preferably from about 14 to about 30nucleotides. The ability to create an antisense or a senseoligonucleotide, based upon a cDNA sequence for a given protein isdescribed in, for example, Stein and Cohen, Cancer Res. 48:2659, 1988and van der Krol et al., BioTechniques 6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acidsequences results in the formation of duplexes that block translation(RNA) or transcription (DNA) by one of several means, including enhanceddegradation of the duplexes, premature termination of transcription ortranslation, or by other means. The antisense oligonucleotides thus maybe used to block expression of hek-L proteins. Antisense or senseoligonucleotides further comprise oligonucleotides having modifiedsugar-phosphodiester backbones (or other sugar linkages, such as thosedescribed in WO 91/06629) and wherein such sugar linkages are resistantto endogenous nucleases. Such oligonucleotides with resistant sugarlinkages are stable in vivo (i.e., capable of resisting enzymaticdegradation) but retain sequence specificity to be able to bind totarget nucleotide sequences. Other examples of sense or antisenseoligonucleotides include those oligonucleotides which are covalentlylinked to organic moieties, such as those described in WO 90/10448, andother moieties that increases affinity of the oligonucleotide for atarget nucleic acid sequence, such as poly-(L-lysine). Further still,intercalating agents, such as ellipticine, and alkylating agents ormetal complexes may be attached to sense or antisense oligonucleotidesto modify binding specificities of the antisense or senseoligonucleotide for the target nucleotide sequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄ -mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by insertion ofthe antisense or sense oligonucleotide into a suitable retroviralvector, then contacting the cell with the retrovirus vector containingthe inserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, the murine retrovirus M-MuLV,N2 (a retrovirus derived from M-MuLV), or the double copy vectorsdesignated DCT5A, DCT5B and DCT5C (see PCT application U.S. 90/02656).

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target nucleic acid sequence by formation ofan oligonucleotide-lipid complex, as described in WO 90/10448. The senseor antisense oligonucleotide-lipid complex is preferably dissociatedwithin the cell by an endogenous lipase.

The following examples are provided to illustrate particular embodimentsand not to limit the scope of the invention.

EXAMPLE 1 Preparation of Soluble hek/FC Fusion Protein

This example describes construction of an expression vector encoding asoluble hek/Fc fusion protein, for use in isolating cDNA clones encodinga hek ligand (hek-L). A DNA and encoded amino acid sequence for humanhek cDNA is presented in Wicks et al. (Proc. Nat'l. Acad. Sci. USA,89:1161, 1992), hereby incorporated by reference. This hek proteincomprises (from N- to C-terminus) an extracellular domain, atransmembrane domain, and a cytoplasmic domain.

Two DNA fragments, one encoding an N-terminal fragment of theextracellular domain of hek and the other encoding a C-terminal fragmentof the hek extracellular domain, were isolated by polymerase chainreactions (PCR) conducted under standard conditions, usingoligonucleotide primers based on the hek nucleotide sequence publishedby Wicks et al., supra. The template for the PCR was cDNA prepared frommRNA isolated from a human T-cell leukemic cell line designatedCCRF-HSB-2 (ATCC CCL-120.1). The PCR products containing the 5' end ofthe hek DNA were digested with SpeI and HindIIIto isolate a DNA fragmentextending from the 5' end of the mature human hek sequence (i.e.,lacking DNA encoding the signal sequence) to a HindIII site found in thehek gene. The PCR products containing the 3' end of the hekextracellular domain DNA were digested with HindIII and ClaI to isolatea fragment extending from the internal HindIII site to a ClaI site justdownstream of the 3' end of the sequence encoding the hek extracellulardomain. The ClaI site is in a multiple cloning site (mcs) introducedjust downstream of the extracellular domain.

DNA encoding a mutein of the Fc region of a human IgG1 antibody wasisolated. This Fc mutein DNA and the polypeptide encoded thereby aredescribed in U.S. patent application Ser. No. 08/097,827, entitled"Novel Cytokine Which is a Ligand for OX40" filed Jul. 23, 1993, whichapplication is hereby incorporated by reference. The mutein DNA wasderived from a native Fc polypeptide-encoding DNA by site-directedmutagenesis conducted essentially as described by Deng and Nickoloff,Anal. Biochem. 200:81 (1992). The amino acid sequence of the Fc muteinpolypeptide is identical to that of the native Fc polypeptide describedin PCT application WO 93/10151, except that amino acid 19 has beenchanged from Leu to Ala, amino acid 20 has been changed from Leu to Glu,and amino acid 22 has been changed from Gly to Ala. This mutein Fcexhibits reduced affinity for immunoglobulin receptors.

A recombinant vector containing the Fc mutein DNA was cleaved with ClaIand NotI, which cleave the vector in a polylinker region immediatelyupstream and downstream, respectively, of the Fc mutein DNA insert. Thedesired Fc mutein-encoding fragment was isolated.

The mutein Fc polypeptide extends from the N-terminal hinge region tothe native C-terminus, i.e., is an essentially full-length antibody Fcregion. Fragments of Fc regions, e.g., those that are truncated at theC-terminal end, also may be employed. The fragments preferably containmultiple cysteine residues (at least the cysteine residues in the hingereaction) to permit interchain disulfide bonds to form between the Fcpolypeptide portions of two separate hek/Fc fusion proteins, creatingdimers.

A mammalian expression vector designated SMAG4 was cleaved with SpeI andNotI. The SMAG4 vector comprises a murine interleukin-7 signalpeptide-encoding sequence (described in U.S. Pat. No. 4,965,195)inserted into the mammalian high expression vector pDC201 (described inSims et al., Science 241:585, 1988, and in PCT application WO 89/03884),which is also capable of replication in E. coli. SpeI cleaves the vectorimmediately downstream of the IL-7 signal peptide-encoding sequence.NotI cleaves approximately 155 bp downstream of the SpeI site in amultiple cloning site of the vector. The large SpeI/NotI fragmentcontaining the vector sequences and the IL-7 signal peptide-encoding DNAwas isolated.

A four-way ligation was conducted to insert the two hek-encoding DNAfragments and the Fc mutein-encoding DNA fragment described above intothe SpeI/NotI cleaved SMAG4 expression vector. E. coli cells weretransfected with the ligation mixture and the desired recombinant vectorwas isolated therefrom. The isolated vector encodes a fusion proteincomprising (from N- to C-terminus) the murine IL-7 signal peptide, thehek extracellular domain, four amino acids encoded by the introducedmcs, and the Fc mutein.

The expression vector was then co-transfected with plasmid pSV3.NEO intoCV1/EBNA cells. The CV1/EBNA cell line (ATCC CRL 10478) was derived froma monkey kidney cell line as described in McMahan et al. (EMBO J.,10:2821, 1991). Vector pSV3.NEO expresses SV40 T-antigen, which is notproduced by the host cells. The pSV3.NEO vector is similar to pSV3(Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78:2072, 1981), butadditionally contains a neomycin resistance gene. The transformed cellswere cultivated to allow transient expression of the fusion protein,which is secreted into the culture medium via the murine IL-7 signalpeptide. The fusion protein was purified on a protein A Sepharosecolumn, eluted, and used to screen cells for the ability to bind thehek/Fc protein, as described in Examples 2 and 3.

EXAMPLE 2 Screening Cells for hek/Fc Binding

Various cell types were screened for the ability to bind hek/Fc, toidentify candidate cell types useful as nucleic acid sources in anattempt to clone a hek ligand. Cells were incubated with the hek/Fcprotein prepared in Example 1, followed by a biotinylated mouseanti-human Fc antibody, followed by streptavidin-phycoerythrin (BectonDickinson). Cells were washed between steps to remove unbound reagents.The biotinylated antibody was purchased from Jackson ImmunoresearchLaboratories, West Grove, Pa. This antibody showed minimal binding to Fcproteins bound to the Fcγ receptor. Streptavidin binds to the biotinmolecule attached to the anti-human Fc antibody, which in turn binds tothe Fc portion of the hek/Fc fusion protein. Phycoerythrin is afluorescent phycobiliprotein which serves as a detectable label. Thelevel of fluorescence signal was measured for each cell type using aFACScan® flow cytometer (Becton Dickinson).

A human T-cell leukemia cell line designated CCRF-HSB-2 (ATCC CCL 120.1)was positive for hek/Fc binding. CCRF-HSB-2 cells were sorted four timesby FACS (fluorescence-activated cell sorting) to derive cells expressinghigher levels of hek/Fc-binding protein.

EXAMPLE 3 Isolation of Hek Ligand cDNA

mRNA was isolated from the 4X sorted CCRF-HSB-2 cells anddouble-stranded cDNA was synthesized on the mRNA template by standardtechniques. A cDNA library was prepared by ligating the cDNA into theBglII site of pDC410 by an adapter method similar to that described byHaymerle et al. (Nucl. Acids Res. 14:8615, 1986). pDC410 is anexpression vector similar to pDC406 (McMahan et al., EMBO J., 10:2821,1991). In pDC410, the EBV origin of replication of pDC406 is replaced byDNA encoding the SV40 large T antigen (driven from an SV40 promoter).The pDC410 multiple cloning site (mcs) differs from that of pDC406 inthat it contains additional restriction sites and three stop codons (onein each reading frame). A T7 polymerase promoter downstream of the mcsfacilitates sequencing of DNA inserted into the mcs.

E. coli strain DH5α cells transfected with the cDNA library in pDC410were plated to provide approximately 2000 colonies per plate. Colonieswere scraped from each plate, pooled, and plasmid DNA prepared from eachpool. The pooled DNA representing about 2000 colonies was then used totransfect a sub-confluent layer of CV1/EBNA-1 cells (described inExample 1). Prior to transfection, the CV1/EBNA-1 cells were maintainedin complete medium (Dulbecco's modified Eagle's media (DMEM) containing10% (v/v) fetal calf serum (FCS), 50 U/ml penicillin, 50 U/mlstreptomycin, 2 mM L-glutamine) and were plated at a density of 2×10⁵cells/well on single-well chambered slides (Lab-Tek). Transfectioninvolved DEAE-dextran followed by chloroquine treatment, similar to theprocedure described by Luthman et al., Nucl. Acids Res. 11:1295, 1983)and McCutchan et al., J. Natl. Cancer Inst. 41:351, 1986). Briefly,slides were pretreated with 1 ml human fibronectin (10 μg/ml in PBS) for30 minutes followed by 1 wash with PBS. Media was removed from theadherent cell layer and replaced with 1.5 ml complete medium containing66.6 μM chloroquine sulfate. 0.2 mls of DNA solution (2 μg DNA, 0.5mg/ml DEAE-dextran in complete medium containing chloroquine) was thenadded to the cells and incubated for 5 hours. Following the incubation,the media was removed and the cells shocked by addition of completemedium containing 10% DMSO for 2.5 to 20 minutes followed by replacementof the solution with fresh complete medium. The cells were cultured for2 to 3 days to permit transient expression of the inserted sequences.

Transfected monolayers of CV1/EBNA-1 cells were assayed for expressionof hek-L by the slide autoradiography procedure of Gearing et al. (EMBOJ. 8:3667, 1989), as follows. Mouse anti-human Fc antibody (JacksonImmunoresearch Laboratories, West Grove, Pa.) was radioiodinated by thechloramine-T method for use in the assay. Briefly, a P6 column wasprepared according to the manufacturer's instructions. In a microfugetube, 10 μg of antibody was dissolved in 10 μl of PBS. 2000 μCi ofcarrier-free Na¹²⁵ I was added and the solution was mixed well. 15 μl ofa freshly prepared solution of chloramine-T (32 μg/ml in 0.05M sodiumphosphate buffer (pH 7.2) was then added and the mixture was incubatedfor 30 minutes at room temperature. The mixture was immediately appliedto the P6 column. The radiolabeled antibody was then eluted from thecolumn by collecting 100-150 μl fractions of eluate. Binding media (RPMI1640 containing 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20mM Hepes pH 7.2) was added to peak fractions to bring the total volumeof each fraction to 2 ml. Radioiodination yielded specific activities inthe range of 5-10×10¹⁵ cpm/mmol protein.

Slide autoradiography was conducted as follows. Transferred CV1/EBNA-1cells (adhered to chambered slides) were washed once with binding mediumwith nonfat dry milk (BM-NFDM) (RPMI medium 1640 containing 25 mg/mlbovine serum albumin (BSA), 2 mg/ml sodium azide, 20 mM HEPES, pH 7.2,and 50 mg/ml nonfat dry milk). Cells were then incubated with hek/Fc(prepared in Example 1) in BM-NFDM (1 μg/ml) for 1 hour at roomtemperature. After incubation, the cell monolayers in the chamberedslides were washed three times with BM-NFDM to remove unbound hek/Fcfusion protein and then incubated with 40 ng/ml ¹²⁵ I-mouse anti-humanFc antibody (a 1:50 dilution) for 1 hour at room temperature. The cellswere washed three times with BM-NFDM, followed by 2 washes withphosphate-buffered saline (PBS) to remove unbound ¹²⁵ I-mouse anti-humanFc antibody. The cells were fixed by incubating for 30 minutes at roomtemperature in 2.5% glutaraldehyde in PBS, pH 7.3, washed twice in PBSand air dried. The chambered slides containing the cells were exposed ona Phophorimager (Molecular Dynamics) overnight, then dipped in KodakGTNB-2 photographic emulsion (6x dilution in water) and exposed in thedark for 3-5 days at 4° C. in a light proof box. The slides were thendeveloped for approximately 4 minutes in Kodak D19 developer (40 g/500ml water), rinsed in water and fixed in Agfa G433C fixer. The slideswere individually examined with a microscope at 25-40× magnification andpositive cells expressing hek-L were identified by the presence ofautoradiographic silver grains against a light background.

Approximately 300,000 cDNAs were screened in pools of approximately2,000 cDNAs to identify transfectant pools showing multiple cellspositive for hek/Fc binding. A positive pool was then partitioned intopools of 500 and again screened by slide autoradiography. A positivepool was identified, partitioned into pools of 100, and screened by thesame procedure. Individual colonies from a positive pool were screeneduntil a single clone (clone #A2) that directed synthesis of a surfaceprotein with detectable hek/Fc binding activity was identified. A secondclone, designated C6, was isolated from a different positive pool. ThecDNA inserts of both clones were sequenced.

The nucleotide and encoded amino acid sequences of the coding region ofthe human hek ligand cDNA of clone A2 are presented in SEQ ID NO:1 andSEQ ID NO:2. The protein comprises an N-terminal signal peptide (aminoacids -19 to -1), an extracellular domain (amino acids 1-202) and aC-terminal domain containing a hydrophobic region (amino acids 203-219).

Human hek-L cDNA was excised from clone A2 by digestion with BglII. Theexcised cDNA was cloned into the BamHI site (in the multiple cloningsite) of pBLUESCRIPT® SK(-) (Stratagene Cloning Systems, La Jolla,Calif.). The resulting vector (designated A2/pBS) in E. coli DH5α cellswas deposited with the American Type Culture Collection, Rockville, Md.,USA (ATCC) on Aug. 11,1993, and assigned accession number ATCC 69384.The deposit was made under the terms of the Budapest Treaty.

The nucleotide and encoded amino acid sequences of the coding region ofthe human hek lieand cDNA of clone C6 are presented in SEQ ID NO:3 andSEQ ID NO:4. The protein comprises an N-terminal signal peptide (aminoacids -22 to -1), an extracellular domain (amino acids 1-160), and aC-terminal domain containing a hydrophobic region (amino acids 161-179).

Human hek-L cDNA was excised from clone C6 by digestion with BglII andinserted into the BamHI site of pBLUESCRIPT® SK(-). The resulting vector(designated C6/pBS) in E. coli DH5α cells was deposited with theAmerican Type Culture Collection, Rockville, Md., USA (ATCC) on Aug. 25,1993 and assigned accession number ATCC 69395. The deposit was madeunder the terms of the Budapest Treaty.

The above-described boundaries of the domains of the hek-L proteins areapproximate, as will be appreciated by the skilled artisan. For example,regarding the hek-L encoded by clone A2, the signal peptide most likelycomprises amino acids -19 to -1, but it is possible that amino acids -19through 3 constitute the signal peptide. Thus, cleavage of the signalpeptide may occur between amino acids -1 and 1 or between amino acids 3and 4, or at both positions. The terms "signal peptide" and "matureprotein" as used herein in reference to the clone A2-encoded hek-L areunderstood to encompass both alternatives, as well as other alternativesdescribed herein for hek-L polypeptides encoded by clone A2 or C6.

The hek-L proteins encoded by clones A2 and C6 have been found to beattached to the cell membrane via glycosyl-phosphatidylinositol (GPI)groups. The hydrophobic domains thus are believed to contain signals forGPI anchoring. Processing of proteins to effect GPI anchoring, whichincludes cleavage of C-terminal sequences, is described above.

EXAMPLE 4 Monoclonal Antibodies to hek-L

This example illustrates the preparation of monoclonal antibodies tohek-L. hek-L is expressed in mammalian host cells such as COS-7 orCV-1/EBNA-1 cells and purified using hek/Fc affinity chromatography.Purified hek-L (or a fragment thereof such as the extracellular domainor immunogenic peptide fragments thereof) can be used to generatemonoclonal antibodies against hek-L using conventional techniques, forexample, those techniques described in U.S. Pat. No. 4,411,993. Briefly,mice are immunized with hek-L as an immunogen emulsified in completeFreund's adjuvant, and injected in amounts ranging from 10-100 μgsubcutaneously or intraperitoneally. Ten to twelve days later, theimmunized animals are boosted with additional hek-L emulsified inincomplete Freund's adjuvant. Mice are periodically boosted thereafteron a weekly to bi-weekly immunization schedule. Serum samples areperiodically taken by retro-orbital bleeding or tail-tip excision fortesting by dot blot assay or ELISA (Enzyme-Linked Immunosorbent Assay),for hek-L antibodies.

Following detection of an appropriate antibody titer, positive animalsare provided one last intravenous injection of hek-L in saline. Three tofour days later, the animals are sacrificed, spleen cells harvested, andspleen cells are fused to a murine myeloma cell line, e.g., NS1 orpreferably P3×63Ag8.653 (ATCC CRL 1580). Fusions generate hybridomacells, which are plated in multiple microtiter plates in a HAT(hypoxanthine, aminopterin and thymidine) selective medium to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

The hybridoma cells are screened by ELISA for reactivity againstpurified hek-L by adaptations of the techniques disclosed in Engvall etal., Immunochem. 8:871,1971 and in U.S. Pat. No. 4,703,004. A preferredscreening technique is the antibody capture technique described inBeckmann et al., (J. Immunol. 144:4212, 1990). Positive hybridoma cellscan be injected intraperitoneally into syngeneic BALB/c mice to produceascites containing high concentrations of anti-hek-L monoclonalantibodies. Alternatively, hybridoma cells can be grown in vitro inflasks or roller bottles by various techniques. Monoclonal antibodiesproduced in mouse ascites can be purified by ammonium sulfateprecipitation, followed by gel exclusion chromatography. Alternatively,affinity chromatography based upon binding of antibody to protein A orprotein G can also be used, as can affinity chromatography based uponbinding to hek-L.

EXAMPLE 5 Binding Studies

The affinity of the hek ligands of the present invention for hek wasdetermined. The hek ligands were also found to bind to a receptortyrosine kinase known as elk, which is distinct from hek. The ability ofcertain other proteins to bind to hek or elk was investigated as well.These studies were conducted as follows.

a) Hek Binding

CV1-EBNA-1 cells (described in example 1) in 12-well plates (2.5×10⁵cells/well) were transfected with clone A2 or C6 (clone A2 cDNA or cloneC6 cDNA in expression vector pDC410, as described in Example 3). Thetransfected cells were cultured for two days to permit expression of thehek-L proteins, which were retained on the cell membrane. The cells thenwere washed with BM-NFDM (see Example 3) and incubated with variousconcentrations of the human hek/Fc fusion protein prepared in Example 1,for 1 hour at room temperature. Subsequently, cells were washed andincubated with the ¹²⁵ I-labeled mouse anti-human IgG antibody preparedin Example 3 (40 ng/ml) in binding medium with gentle agitation for 1hour at 37° C. Cells then were harvested by trypsinization. In allassays, non-specific binding of ¹²⁵ I antibody was assayed in theabsence of hek/Fc as well as in the presence of hek/Fc and a 200-foldmolar excess of unlabeled mouse anti-human IgG antibody. Free andcell-bound ¹²⁵ I-antibody were quantified on a Packard AutogammaCounter. Affinity calculations (Scatchard, Ann. N.Y. Acad. Sci. 51:660,1949) were generated on RS/1 (BBN Software, Boston, Mass.) run on aMicrovax computer. CV1-EBNA-1 cells transfected with an "empty" pDC410vector were included in the same binding study as a control.

Hek/Fc binding to CV1-EBNA-1 cells expressing two different recombinantproteins, human elk-L and human B61, also was analyzed in theabove-described experiment. The cells were transfected with elk ligand(elk-L) or B61 cDNA in vector pDC410. The expressed proteins were cellmembrane bound.

Elk-L binds to a receptor known as elk, which, like hek, is a member ofthe eph/elk family of receptor tyrosine kinases (see the "Background ofthe Invention" section above). Elk-L was included in this study toinvestigate whether or not it would bind to a different receptor of thesame family (i.e., hek). The protein known as B61 has been identified asthe product of a novel immediate-early response gene induced by TNF inhuman umbilical vein endothelial cells (Holzman et al., Mol. Cell. Biol.10:5830, 1990). B61 was included in the study because of its degree ofhomology to elk-L (33% identity at the amino acid level).

The nucleotide sequence of isolated B61 cDNA and the amino acid sequenceencoded thereby are presented in Holzman et al., supra, herebyincorporated by reference in its entirety. Methods for producing andrecovering B61 are also described, along with certain structuralcharacteristics and properties of the protein. Nucleotide and encodedamino acid sequences for elk-L cDNA are described in copending U.S.application Ser. No. 07/977,693, filed Nov. 13, 1992, and herebyincorporated by reference in its entirety. Production and purificationof elk-L are also described, and certain functional domains of theprotein are identified. E. coli DH5α cells transformed with human elk-LcDNA inserted into the SmaI site (in the mcs) of cloning vectorpBLUESCRIPT® SK (Stratagene, La Jolla, Calif.) was deposited as ATCC69085 on Oct. 9, 1992.

The results of the study were as follows:

                  TABLE I                                                         ______________________________________                                                     Binding affinity for hek/Fc (K.sub.a)                            ______________________________________                                        pDC410         --                                                             B61            5.5 × 10.sup.7 M.sup.-1                                  elk-L          2.3 × 10.sup.7 M.sup.-1 ; 2.9 × 10.sup.6                          M.sup.-1                                                       hek-L A2       2.0 × 10.sup.8 M.sup.-1                                  hek-L C6       2.0 × 10.sup.8 M.sup.-1                                  ______________________________________                                    

The empty vector exhibited no detectable hek/Fc binding. B61 boundhek/Fc with relatively moderate affinity, exhibiting a single affinityclass of binding. The binding of hek/Fc to elk-L resulted in a biphasicpattern, indicating two lower-affinity binding components (affinityconstants 2.3×10⁷ M⁻¹ and 2.9×10⁶ M⁻¹). The affinities of the two hek-Lproteins for hek/Fc were equivalent and relatively high.

b) Elk Binding

The binding assay described above was repeated, substituting a solublerat elk/Fc fusion protein for the hek/Fc fusion protein. Nucleotide andencoded amino acid sequences for rat elk are presented in Lhotak et al.(Mol. Cell. Biol. 11:2496, 1991). The elk/Fc fusion protein comprisedthe extracellular domain of elk fused to a native Fc region polypeptidederived from a human IgG1 antibody. The indirect assay (using unlabeledelk/Fc and radioiodinated mouse anti-human IgG antibody) was employedbecause direct radiolabeling of elk/Fc inactivated the bindingspecificity thereof.

The results of the study were as follows:

                  TABLE II                                                        ______________________________________                                                     Binding affinity for elk/Fc (K.sub.a)                            ______________________________________                                        B61            2.3 × 10.sup.8 M.sup.-1 ; 7.0 × 10.sup.7                          M.sup.-1                                                       elk-L          1.08 × 10.sup.9 M.sup.-1                                 hek-L A2       2.7 × 10.sup.8 M.sup.-1 ; 3.5 × 10.sup.7                          M.sup.-1                                                       hek-L C6       1.3 × 10.sup.8 M.sup.-1 ; 5.4 × 10.sup.7                          M.sup.-1                                                       ______________________________________                                    

A biphasic pattern of elk/Fc binding was observed for B61 with K_(a) sof 2.3×10⁸ M⁻¹ and 7.0×10⁷ M⁻¹. The affinity constant (K_(a)) shown forelk/Fc binding to transfected cells expressing elk-L matches well withthose observed for binding of elk/Fc to the native ligand expressed onvarious rat neural cell lines. A biphasic pattern of elk/Fc binding isseen for both hek ligands.

EXAMPLE 6 Homology

The homology of the full length human elk-L, B61, hek ligand A2, and hekligand C6 proteins (described in Example 5) for one another at the aminoacid level are presented in Table III:

                  TABLE III                                                       ______________________________________                                                       % amino acid identity                                                         elk-L                                                                              B61      A2    C6                                         ______________________________________                                        % amino acid elk-L          33     28  32                                     similarity   B61     51            40  37                                                  A2      48     63         38                                                  C6      50     55     57                                         ______________________________________                                    

The percent identity of the DNA sequences are presented in Table IV:

                  TABLE IV                                                        ______________________________________                                                % DNA identity                                                                elk-L                                                                              B61          A2     C6                                           ______________________________________                                        elk-L            44.0         40.7 43.7                                       B61                           48.9 51.5                                       A2                                 47.3                                       C6                                                                            ______________________________________                                    

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1037 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: hek-L A2                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 83..799                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: sig_peptide                                                     (B) LOCATION: 83..139                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat_peptide                                                     (B) LOCATION: 140..796                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGATCTTGGAACGAGACGACCTGCTGGAGAAGCCGGGAGCGCGGGGCTCAGTCGGGGGGC60                GGCGGCGGCGGCGGCTCCGGGGATGGCGGCGGCTCCGCTGCTGCTGCTGCTG112                       MetAlaAlaAlaProLeuLeuLeuLeuLeu                                                19-15- 10                                                                     CTGCTCGTGCCCGTGCCGCTGCTGCCGCTGCTGGCCCAAGGGCCCGGA160                           LeuLeuValProValProLeuLeuProLeuLeuAlaGlnGlyProGly                              515                                                                           GGGGCGCTGGGAAACCGGCATGCGGTGTACTGGAACAGCTCCAACCAG208                           GlyAlaLeuGlyAsnArgHisAlaValTyrTrpAsnSerSerAsnGln                              101520                                                                        CACCTGCGGCGAGAGGGCTACACCGTGCAGGTGAACGTGAACGACTAT256                           HisLeuArgArgGluGlyTyrThrValGlnValAsnValAsnAspTyr                              253035                                                                        CTGGATATTTACTGCCCGCACTACAACAGCTCGGGGGTGGGCCCCGGG304                           LeuAspIleTyrCysProHisTyrAsnSerSerGlyValGlyProGly                              40455055                                                                      GCGGGACCGGGGCCCGGAGGCGGGGCAGAGCAGTACGTGCTGTACATG352                           AlaGlyProGlyProGlyGlyGlyAlaGluGlnTyrValLeuTyrMet                              606570                                                                        GTGAGCCGCAACGGCTACCGCACCTGCAACGCCAGCCAGGGCTTCAAG400                           ValSerArgAsnGlyTyrArgThrCysAsnAlaSerGlnGlyPheLys                              758085                                                                        CGCTGGGAGTGCAACCGGCCGCACGCCCCGCACAGCCCCATCAAGTTC448                           ArgTrpGluCysAsnArgProHisAlaProHisSerProIleLysPhe                              9095100                                                                       TCGGAGAAGTTCCAGCGCTACAGCGCCTTCTCTCTGGGCTACGAGTTC496                           SerGluLysPheGlnArgTyrSerAlaPheSerLeuGlyTyrGluPhe                              105110115                                                                     CACGCCGGCCACGAGTACTACTACATCTCCACGCCCACTCACAACCTG544                           HisAlaGlyHisGluTyrTyrTyrIleSerThrProThrHisAsnLeu                              120125130135                                                                  CACTGGAAGTGTCTGAGGATGAAGGTGTTCGTCTGCTGCGCCTCCACA592                           HisTrpLysCysLeuArgMetLysValPheValCysCysAlaSerThr                              140145150                                                                     TCGCACTCCGGGGAGAAGCCGGTCCCCACTCTCCCCCAGTTCACCATG640                           SerHisSerGlyGluLysProValProThrLeuProGlnPheThrMet                              155160165                                                                     GGCCCCAATGTGAAGATCAACGTGCTGGAAGACTTTGAGGGAGAGAAC688                           GlyProAsnValLysIleAsnValLeuGluAspPheGluGlyGluAsn                              170175180                                                                     CCTCAGGTGCCCAAGCTTGAGAAGAGCATCAGCGGGACCAGCCCCAAA736                           ProGlnValProLysLeuGluLysSerIleSerGlyThrSerProLys                              185190195                                                                     CGGGAACACCTGCCCCTGGCCGTGGGCATCGCCTTCTTCCTCATGACG784                           ArgGluHisLeuProLeuAlaValGlyIleAlaPhePheLeuMetThr                              200205210215                                                                  TTCTTGGCCTCCTAGCTCTGCCCCCTCCCCTGGGGGGGGAGAGATGGGGCGG836                       PheLeuAlaSer                                                                  220                                                                           GGCTTGGAAGGAGCAGGGAGCCTTTGGCCTCTCCAAGGGAAGCCTAGTGGGCCTAGACCC896               CTCCTCCCATGGCTAGAAGTGGGGCCTGCACCATACATCTGTGTCCGCCCCCTCTACCCC956               TTCCCCCCACGTAGGGCACTGTAGTGGACCAAGCACGGGGACAGCCATGGGTCCCGAGCA1016              GGTCGTCTCGTTCCAAGATCC1037                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 238 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaAlaAlaProLeuLeuLeuLeuLeuLeuLeuValProValPro                              19- 15-10-5                                                                   LeuLeuProLeuLeuAlaGlnGlyProGlyGlyAlaLeuGlyAsnArg                              1510                                                                          HisAlaValTyrTrpAsnSerSerAsnGlnHisLeuArgArgGluGly                              152025                                                                        TyrThrValGlnValAsnValAsnAspTyrLeuAspIleTyrCysPro                              30354045                                                                      HisTyrAsnSerSerGlyValGlyProGlyAlaGlyProGlyProGly                              505560                                                                        GlyGlyAlaGluGlnTyrValLeuTyrMetValSerArgAsnGlyTyr                              657075                                                                        ArgThrCysAsnAlaSerGlnGlyPheLysArgTrpGluCysAsnArg                              808590                                                                        ProHisAlaProHisSerProIleLysPheSerGluLysPheGlnArg                              95100105                                                                      TyrSerAlaPheSerLeuGlyTyrGluPheHisAlaGlyHisGluTyr                              110115120125                                                                  TyrTyrIleSerThrProThrHisAsnLeuHisTrpLysCysLeuArg                              130135140                                                                     MetLysValPheValCysCysAlaSerThrSerHisSerGlyGluLys                              145150155                                                                     ProValProThrLeuProGlnPheThrMetGlyProAsnValLysIle                              160165170                                                                     AsnValLeuGluAspPheGluGlyGluAsnProGlnValProLysLeu                              175180185                                                                     GluLysSerIleSerGlyThrSerProLysArgGluHisLeuProLeu                              190195200205                                                                  AlaValGlyIleAlaPhePheLeuMetThrPheLeuAlaSer                                    210215                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 636 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA to mRNA                                              (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: hek-L C6                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: mat_peptide                                                     (B) LOCATION: 94..630                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 28..633                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: sig_peptide                                                     (B) LOCATION: 28..93                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GCCAGACCAAACCGGACCTCGGGGGCGATGCGGCTGCTGCCCCTGCTGCGG51                         MetArgLeuLeuProLeuLeuArg                                                      22-20-15                                                                      ACTGTCCTCTGGGCCGCGTTCCTCGGCTCCCCTCTGCGCGGGGGCTCC99                            ThrValLeuTrpAlaAlaPheLeuGlySerProLeuArgGlyGlySer                              10-51                                                                         AGCCTCCGCCACGTAGTCTACTGGAACTCCAGTAACCCCAGGTTGCTT147                           SerLeuArgHisValValTyrTrpAsnSerSerAsnProArgLeuLeu                              51015                                                                         CGAGGAGACGCCGTGGTGGAGCTGGGCCTCAACGATTACCTAGACATT195                           ArgGlyAspAlaValValGluLeuGlyLeuAsnAspTyrLeuAspIle                              202530                                                                        GTCTGCCCCCACTACGAAGGCCCAGGGCCCCCTGAGGGCCCCGAGACG243                           ValCysProHisTyrGluGlyProGlyProProGluGlyProGluThr                              35404550                                                                      TTTGCTTTGTACATGGTGGACTGGCCAGGCTATGAGTCCTGCCAGGCA291                           PheAlaLeuTyrMetValAspTrpProGlyTyrGluSerCysGlnAla                              556065                                                                        GAGGGCCCCCGGGCCTACAAGCGCTGGGTGTGCTCCCTGCCCTTTGGC339                           GluGlyProArgAlaTyrLysArgTrpValCysSerLeuProPheGly                              707580                                                                        CATGTTCAATTCTCAGAGAAGATTCAGCGCTTCACACCTTTCTCCCTC387                           HisValGlnPheSerGluLysIleGlnArgPheThrProPheSerLeu                              859095                                                                        GGCTTTGAGTTCTTACCTGGAGAGACTTACTACTACATCTCGGTGCCC435                           GlyPheGluPheLeuProGlyGluThrTyrTyrTyrIleSerValPro                              100105110                                                                     ACTCCAGAGAGTTCTGGCCAGTGCTTGAGGCTCCAGGTGTCTGTCTGC483                           ThrProGluSerSerGlyGlnCysLeuArgLeuGlnValSerValCys                              115120125130                                                                  TGCAAGGAGAGGAAGTCTGAGTCAGCCCATCCTGTTGGGAGCCCTGGA531                           CysLysGluArgLysSerGluSerAlaHisProValGlySerProGly                              135140145                                                                     GAGAGTGGCACATCAGGGTGGCGAGGGGGGGACACTCCCAGCCCCCTC579                           GluSerGlyThrSerGlyTrpArgGlyGlyAspThrProSerProLeu                              150155160                                                                     TGTCTCTTGCTATTACTGCTGCTTCTGATTCTTCGTCTTCTGCGAATT627                           CysLeuLeuLeuLeuLeuLeuLeuLeuIleLeuArgLeuLeuArgIle                              165170175                                                                     CTGTGAGCC636                                                                  Leu                                                                           180                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 201 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetArgLeuLeuProLeuLeuArgThrValLeuTrpAlaAlaPheLeu                              22-20-15-10                                                                   GlySerProLeuArgGlyGlySerSerLeuArgHisValValTyrTrp                              51510                                                                         AsnSerSerAsnProArgLeuLeuArgGlyAspAlaValValGluLeu                              152025                                                                        GlyLeuAsnAspTyrLeuAspIleValCysProHisTyrGluGlyPro                              303540                                                                        GlyProProGluGlyProGluThrPheAlaLeuTyrMetValAspTrp                              455055                                                                        ProGlyTyrGluSerCysGlnAlaGluGlyProArgAlaTyrLysArg                              606570                                                                        TrpValCysSerLeuProPheGlyHisValGlnPheSerGluLysIle                              75808590                                                                      GlnArgPheThrProPheSerLeuGlyPheGluPheLeuProGlyGlu                              95100105                                                                      ThrTyrTyrTyrIleSerValProThrProGluSerSerGlyGlnCys                              110115120                                                                     LeuArgLeuGlnValSerValCysCysLysGluArgLysSerGluSer                              125130135                                                                     AlaHisProValGlySerProGlyGluSerGlyThrSerGlyTrpArg                              140145150                                                                     GlyGlyAspThrProSerProLeuCysLeuLeuLeuLeuLeuLeuLeu                              155160165170                                                                  LeuIleLeuArgLeuLeuArgIleLeu                                                   175                                                                           __________________________________________________________________________

What is claimed is:
 1. A purified hek ligand (hek-L) protein that bindshek, wherein said hek-L protein is a fragment of the hek-L polypeptidepresented in SEQ ID NO:2, wherein said fragment binds hek.
 2. Acomposition comprising a hek-L polypeptide according to claim 1, and aphysiologically acceptable carrier, diluent, or excipient.
 3. A purifiedhek-L polypeptide comprising an amino acid sequence selected from thegroup consisting of amino acids 1-202 and 1-219 of SEQ ID NO:2.
 4. Apurified hek ligand (hek-L) protein that binds hek, wherein said hek-Lprotein is a fragment of the hek-L polypeptide presented in SEQ ID NO:4,wherein said fragment binds hek.
 5. A composition comprising a hek-Lpolypeptide according to claim 4, and a physiologically acceptablecarrier, diluent, or excipient.
 6. A purified hek-L polypeptidecomprising an amino acid sequence selected from the group consisting ofamino acids 1-160 and 1-179 of SEQ ID NO:4.
 7. A purified hek-L proteincomprising an amino acid sequence that is at least 90% identical to asequence selected from the group consisting of amino acids 1-219 of SEQID NO:2 and amino acids 1-179 of SEQ ID NO:4, wherein said hek-L proteinbinds hek.
 8. A hek-L protein according to claim 7, wherein said hek-Lprotein comprises an amino acid sequence selected from the groupconsisting of amino acids 1-219 of SEQ ID NO:2 and amino acids 1-179 ofSEQ ID NO:4.
 9. A composition comprising a hek-L polypeptide accordingto claim 7, and a physiologically acceptable carrier, diluent, orexcipient.
 10. A purified hek-L polypeptide that binds hek, wherein saidhek-L polypeptide comprises an amino acid sequence selected from thegroup consisting of:a) amino acids z through x of SEQ ID NO:2, wherein zis an amino acid in positions 1 to 12 of SEQ ID NO:2 and x is an aminoacid in positions 193 to 219 of SEQ ID NO:2; and b) amino acids z'through y of SEQ ID NO:4, wherein z' is an amino acid in positions 1 to4 of SEQ ID NO:4 and y is an amino acid in positions 147 to 179 of SEQID NO:4.
 11. A purified hek-L polypeptide according to claim 10, whereinz is the amino acid in position 1 of SEQ ID NO:2, and z' is the aminoacid in position 1 of SEQ ID NO:4.
 12. A hek-L polypeptide according toclaim 10, wherein x is an amino acid in any of positions 193 to 202 ofSEQ ID NO:2, and y is an amino acid in any of positions 147 to 160 ofSEQ ID NO:4.
 13. A composition comprising a hek-L polypeptide accordingto claim 12, and a physiologically acceptable carrier, diluent, orexcipient.
 14. A hek-L protein encoded by the hek-L cDNA insert of therecombinant vector contained in transformed cells selected from thegroup consisting of transformed cells deposited as ATCC 69384 andtransformed cells deposited as ATCC
 69395. 15. A fusion proteincomprising a hek-L polypeptide that binds hek, and an antibody-derivedpolypeptide, wherein said hek-L polypeptide comprises an amino acidsequence that is at least 90% identical to a sequence selected from thegroup consisting of amino acids 1-202 of SEQ ID NO:2 and amino acids1-160 of SEQ ID NO:4.
 16. A fusion protein according to claim 15,wherein said antibody-derived polypeptide is an Fc polypeptide.
 17. Ahek-L polypeptide comprising conservative substitution(s) in an aminoacid sequence selected from the group consisting of:(a) amino acids1-202 of SEQ ID NO:2, (b) amino acids 1-219 of SEQ ID NO:2, (c) aminoacids 1-160 of SEQ ID NO:4, and (d) amino acids 1-179 of SEQ ID NO:4,wherein said hek-L polypeptide binds hek; wherein, apart from saidconservative substitution(s), the amino acid sequence of said hek-Lpolypeptide is identical to the sequence presented in (a), (b), (c), or(d); wherein the amino acid sequence of said hek-L polypeptide is atleast 90% identical to the sequence presented in (a), (b), (c), or (d).18. A hek-L polypeptide comprising conservative substitution(s) in anamino acid sequence selected from the group consisting of:a) amino acidsz through x of SEQ ID NO:2, wherein z is an amino acid in positions 1 to12 of SEQ ID NO:2 and x is an amino acid in positions 193 to 219 of SEQID NO:2; and b) amino acids z' through y of SEQ ID NO:4, wherein z' isan amino acid in positions 1 to 4 of SEQ ID NO:4 and y is an amino acidin positions 147 to 179 of SEQ ID NO:4, wherein said hek-L polypeptidebinds hek; wherein, apart from said conservative substitution(s), theamino acid sequence of said hek-L polypeptide is identical to thesequence presented in (a) or (b); wherein the amino acid sequence ofsaid hek-L polypeptide is at least 90% identical to the sequencepresented in (a) or (b).
 19. An oligomer comprising from two to foursoluble hek-L polypeptides, wherein said soluble hek-L polypeptides eachcomprise an amino acid sequence selected from the group consisting of:a)amino acids z through x' of SEQ ID NO:2, wherein z is an amino acid inpositions 1 to 12 of SEQ ID NO:2 and x' is an amino acid in positions193 to 202 of SEQ ID NO:2; and b) amino acids z' through y' of SEQ IDNO:4, wherein z' is an amino acid in positions 1 to 4 of SEQ ID NO:4 andy' is an amino acid in positions 147 to 160 of SEQ ID NO:4, wherein saidoligomer binds hek.
 20. A fusion protein comprising a hek-L polypeptideand an Fc polypeptide, wherein said hek-L polypeptide is selected fromthe group consisting of:a) a soluble fragment of the hek-L polypeptidepresented in SEQ ID NO:2, and b) a soluble fragment of the hek-Lpolypeptide presented in SEQ ID NO:4, wherein said fragment binds hek.21. A fusion protein according to claim 20, wherein said hek-Lpolypeptide comprises amino acids z through x' of SEQ ID NO:2, wherein zis an amino acid in any of positions 1 to 12 of SEQ ID NO:2 and x' is anamino acid in any of positions 193 to 202 of SEQ ID NO:2.
 22. A fusionprotein according to claim 20, wherein said hek-L polypeptide comprisesamino acids z' through y' of SEQ ID NO:4, wherein z' is an amino acid inany of positions 1 to 4 of SEQ ID NO:4 and y' is an amino acid in any ofpositions 147 to 160 of SEQ ID NO:4.
 23. An oligomer comprising from twoto four soluble hek-L polypeptides, wherein said soluble hek-Lpolypeptides are each selected from the group consisting of:a) a solublefragment of the hek-L polypeptide presented in SEQ ID NO:2; and b) asoluble fragment of the hek-L polypeptide presented in SEQ ID NO:4;wherein said fragment binds hek.
 24. A composition comprising anoligomer according to claim 23, and a physiologically acceptablecarrier, diluent, or excipient.